Methods for ‘next generation’ (e.g., polony) sequencing typically employed in the art use degenerate oligonucleotides (e.g., octamers and nonamers) and protein ligases to discriminate among oligonucleotides with perfect match at one or more positions relative to mismatched oligonucleotides (see Shendure et al. (2005) Science 309:1728; SOLiD™ (Applied Biosystems, Foster City, Calif.); and Complete Genomics (Mountain View, Calif.)). These technologies can accurately read six nucleotides in from the ligation junction. The current method to increase read-length beyond six bases uses degenerate oligonucleotides containing a cleavable bridged phosphorothioate internucleotide linkage between positions 6 and 7 from the ligation junction (e.g., SOLiD™ (Applied Biosystems)). After ligation to the anchor oligonucleotide bound to the immobilized template, the solid phase is extensively washed to remove free fluor-oligonucleotides and visualized with a digital camera. The oligonucleotides are typically labeled with fluorophores so that a given emission wavelength corresponds with a given base (or bases) (e.g., A, C, G or T) at specific locations in the oligonucleotide bound to the template (e.g., unknown) DNA. Following imaging, the bridged phosphorothioate internucleotide linkage is cleaved, releasing the fluorophores and truncating the oligonucleotide. A subsequent round of degenerate oligonucleotides are ligated, washed and imaged to determine the sequence on positions 7-12 on the template (e.g., using SOLiD™ (Applied Biosystems)).
The main obstacle for increasing read-length in this manner is the synthesis on the oligonucleotides containing the bridged-phosphorothioate internucleotide linkage. These oligonucleotides require commercially unavailable nucleoside phosphoramidites that are expensive to synthesize. In addition, the current technology only allows sequencing in one direction.